PROJECT SUMMARY Protein design offers a promising strategy to alter and optimize proteins for biotechnology, industry, and medicine. Though there are many examples of successful protein designs, reliable design of stable proteins with high biological activity remains challenging. Protein consensus sequence design (using multiple sequence alignment of homologous proteins to identify the sequence of maximal conservation) may provide proteins with high activity as well as high stability, as functionally important residues are likely to be conserved. Several studies have provided anecdotal accounts of stability enhancement using consensus sequence information, although other studies describe mixed results. In this proposal, I aim to generalize the viability of the consensus sequence design strategy to create protein design targets with high stability and biological activity. Motivated by my preliminary results demonstrating stability enhancement and maintenance of biological activity for a small set of consensus protein constructs, I plan to create a larger set of consensus proteins and characterize structure, stability, and function. I have selected ten proteins families for consensus design. I will determine the structures of these target proteins using a combination of NMR and x-ray crystallography, will, determine equilibrium thermodynamic stabilities, and will measure biological activities. As part of the functional characterization, I will examine thermodynamic and kinetic parameters of molecular recognition and enzyme catalysis, quantify consensus protein conformational dynamics, and determine the effects of consensus proteins on cellular fitness. This project will comprehensively evaluate consensus design as an effective protein design strategy, testing the generality to which it may produce proteins of high stability and biological activity. Moreover, the proposed project will answer longstanding unresolved questions on the interrelated nature of protein evolution, stability, dynamics, and function.